Clinical and genetic characterization of pediatric patients with progressive familial intrahepatic cholestasis type 3 (PFIC3): identification of 14 novel ABCB4 variants and review of the literatures

Background Progressive familial intrahepatic cholestasis type 3 (PFIC3) is an autosomal recessive disease caused by pathogenic variants of the gene ABCB4. This study aimed to investigate the ABCB4 genotypic and the clinical phenotypic features of PFIC3 patients. Methods The clinical and molecular genetic data of 13 new pediatric patients with PFIC3 as well as 82 reported ones in the PubMed and CNKI databases were collected and analyzed. Results The 13 new PFIC3 patients included six females and seven males, and the main presentations were hepatomegaly, splenomegaly, jaundice, and pruritus, as well as increased levels of gamma-glutamyl transpeptidase (GGT). Fourteen new ABCB4 variants were detected, including eight diagnosed to be likely-pathogenic and six, pathogenic. Among all the 95 PFIC3 cases, hepatomegaly was observed in 85.3% (81/95), pruritus in 67.4% (64/95), splenomegaly in 52.6% (50/95), jaundice in 48.4% (46/95), portal hypertension in 34.7% (33/95) and GGT elevation in 100% (88/88) of the patients. Positive responses at varied degrees to oral ursodeoxycholic acid (UDCA) treatment were observed in 66.1% (39/59) of the patients, among whom 38.5% (15/39) fully recovered in terms of the laboratory changes. Although the condition remained stable in 53 patients (58.9%, 53/90), the clinical outcomes were not promising in the rest 37 cases (41.1%, 37/90), including 7 died, 27 having undergone while another 3 waiting for liver transplantation. A total of 96 ABCB4 variants were detected in the 95 patients. PFIC3 patients with biallelic null variants exhibited earlier onset ages [10.5 (2, 18) vs. 19 (8, 60) months, p = 0.007], lower UDCA response rate [18.2% (2/11) vs. 77.1% (37/48), p = 0.001], and more unpromising clinical outcomes [80% (12/15) vs. 33.3% (25/75), p = 0.001], compared with those with non-biallelic null variants. Conclusions PFIC3 presented with hepatomegaly, pruritus, splenomegaly and jaundice with increased serum GGT level as a biochemistry hallmark. Although varying degrees of improvement in response to UDCA therapy were observed, 41.1% of PFIC3 patients exhibited unfavorable prognosis. ABCB4 genotypes of biallelic null variants were associated with severer PFIC3 phenotypes. Moreover, the 14 novel variants in this study expanded the ABCB4 mutation spectrum, and provided novel molecular biomarkers for diagnosis of PFIC3 patients. Supplementary Information The online version contains supplementary material available at 10.1186/s13023-022-02597-y.


Introduction
Progressive familial intrahepatic cholestasis (PFIC) included a group of rare autosomal recessive diseases caused by pathogenic variants of the genes encoding proteins related to the formation and transfer of bile acids in the liver [1]. The PFIC patient's onset varied from the neonatal period to early adulthood, which usually developed fibrosis and end-stage liver disease before adulthood [2]. Based on different causative genes, PFIC could be divided into types 1-6, with ATP8B1, ABCB11, ABCB4, TJP2, NR1H4, and MYO5B being the causative gene, respectively [3].
The gene ABCB4 causing PFIC3 (OMIM # 602347) was located on chromosome 7q21, which encoded a liverspecific canalicular transporter, the Multi-Drug Resistant 3 (MDR3) protein [4]. MDR3 translocated phosphatidylcholine from the inner to the outer leaflet of the canalicular membrane, resulting phosphatidylcholine efflux into the bile [5,6]. In the aqueous environment of bile, phospholipids form mixed micelles with cholesterol and bile acids, thereby preventing the formation of cholesterol gallstones and the detergent action of free bile acids which was injurious to cholangiocyte membrane [7,8]. Typical clinical features of PFIC3 included jaundice, pruritus, hepatomegaly, and splenomegaly, which could progress to cirrhosis and liver failure before adulthood. Thus far, due to the lack of pathognomonic clinical symptoms or signs, the definitive diagnosis of PFIC3 relied on the ABCB4 genetic analysis [9].
In the recent years, the clinical application of molecular genetic techniques facilitated the timely diagnosis of PFIC patients, and an increasing number of PFIC3 patients were reported around the world [10][11][12][13][14][15]. However, the molecular and clinical characteristics of this condition, generally as a rare liver disease, remained yet far from being completely understood. This study analyzed the phenotypic and genotypic features of PFIC3 patients by reporting 13 new pediatric patients and reviewing the relevant literatures.

Subjects and ethical approval
The research subjects in this study included 13 pediatric patients including seven males and six females from 12 unrelated families. The clinical data of the 13 patients were collected for analysis, including their ages, genders, history, clinical presentations, laboratory changes, treatment and outcomes. Most the data were collected from the medical record databases in the participating hospitals, with partial data from other hospitals being provided by patients' parents at their referrals to our clinics.
This study was approved by the Medical Ethics Committee of the First Affiliated Hospital, Jinan University, and written informed consents were signed by the parents of all patients before this study.

Genetic analysis
Genomic DNA was obtained from peripheral blood according to standard procedures, and all patients underwent next-generation sequencing (NGS) of the targeted or whole exomes, to explore the underling genetic causes. ABCB4 variants detected were then verified by Sanger sequencing. The sequencing results were aligned with the ABCB4 gene sequence (ENST00000649586.2), which was available at Ensembl Genome Browser (www. ensem bl. org). The variant nomenclature was in agreement with current guidelines of the Human Genome Variation Society (http:// www. hgvs. org/ rec. html).

Pathogenicity evaluation
All the variants were classified according to the American College of Medical Genetics and Genomics (ACMG) standards and guidelines.

Review of the literatures
Electronic databases including PubMed and CNKI (https:// www. cnki. net) were retrieved by using the keywords "ABCB4" and "PFIC3". The genotypic and phenotypic data of the pediatric patients with clear molecular genetic diagnosis and detailed clinical information were collected and analyzed.

Statistical analysis
Data were analyzed with the use of IBM SPSS Statistics 26 software (IBM, Armonk, NY, USA). Normally distributed data were expressed as mean ± SD and then compared using Student's t-test. Data of skewed distribution were presented as the median values (P25, P75), and comparisons were conducted by means of Mann-Whitney U-test. Categorical variables were expressed as percentages, and statistical differences were compared by Chi-Square or Fisher's exact test. Statistical significance was set at p < 0.05. Table 1 summarized the clinical information of the 13 new PFIC3 patients from 12 unrelated families. The ages of symptom onset were 36 (8, 67) months. As the commonest clinical presentation in this cohort, hepatomegaly was observed in 13 patients, followed by splenomegaly in 11, jaundice in seven, pruritus in four cases. The biochemistry hallmark was markedly increased levels of gamma-glutamyl transpeptidase (GGT) in all patients. On the last follow up, two patients demonstrated unfavorable outcomes: one was waiting for liver transplantation due to hepatic decompensation and one had died of liver failure. Nine patients were alive with stable condition, and the rest two lost contact.
The variants c.1801G > A(p.Ala601Thr) and c.3230C > T(p.Thr1077Met) were included in the database gnomeAD with the allele frequencies of 0.007962‰ and 0.1991‰, respectively, while the rest 12 novel variants have neither been reported in any official literatures nor included in any variant databases, to the best of our knowledge. The amino acid sequences of the homologous peptides in a total of 20 primates were aligned comparatively, and the results showed the eight novel missense variants were localized in highly conserved regions of all the 20 primate homologous proteins (Fig. 2).
In the study, six out of the 14 novel variants were classified to be pathogenic, and remaining eight as likely pathogenic, according to the ACMG standards. The relevant evidences were listed in detail in Table 2.

Genotype-phenotype correlation
In this study, the frameshift, nonsense, canonical ± 1 or 2 splicing-site variants and exons deletion were defined as null variants, while missense and non-canonical splicingsite, as non-null variants, according to the ACMG standards. The ABCB4 phenotypes of the 95 PFIC3 patients were categorized into two groups: biallelic null variants (n = 18) and non-biallelic null variants (n = 77). It was found that PFIC3 patients with biallelic null variants exhibited earlier onset ages [10.5 (2, 18)

Discussion
This study described 13 new PFIC3 patients from 12 unrelated families, and among the 23 ABCB4 variants detected, 14 were not reported previously in any official According to the ACMG standards and guidelines, the six novel variants above were all null ABCB4 variants, and was diagnosed to be pathogenic, with the relevant evidences listed in Table 2.
The remaining eight novel missense variants were all absent or included rather rarely included in public databases (PM2), and among them, c.2782A > G(p. Arg928GlyA), c.1645C > T(p.Arg549Cys), c.716C > T(p. Ser239Leu), and c.3230C > T(p.Thr1077Met) proved to be in trans with a previously-reported pathogenic variant by testing parents (PM3) [11,21]. All the novel missense variants cosegregated with the PFIC3 phenotype in this study (PP1). It was well-known that ABCB4 missense variants were a common mechanism for PFIC3 development (PP2) [21,24,27,[32][33][34]. In silico prediction suggested them to be disease-causing/ deleterious/possibly damaging/probably damaging, and with the involved amino acid residues all being highly conserved among 20 primates (PP3). Moreover, the biochemical and clinical presentations of the ten patients were quite specific for PFIC3 (PP4). Although in vitro or in vivo functional analysis was not performed due to technical limitation, the evidences above rendered the eight novel missense variants all "likely pathogenic" and supported the diagnosis of PFIC3 in the patients, since according to the ACMG standards, a variant classified as likely pathogenic typically has sufficient evidence that a health-care provider can use the molecular testing information in clinical  Table 2 Novel ABCB4 variants and the pathogenicity classification According to the ACMG criteria [35]: PVS1, null variant (nonsense, frameshift, canonical ± 1 or 2 ss, etc.) in a gene where loss of function is a known mechanism of disease; PM2, absent from controls (or at extremely low frequency if recessive) absent from controls in 1000 Genomes Project, or gnomAD; PM3, for recessive disorders, detected in trans with a pathogenic variant; PP1, co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease; PP2, missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease; PP3, multiple lines of computational evidence support a deleterious effect on the gene or gene product; PP4, patient's phenotype or family history is highly specific for a disease with a single genetic etiology; -, not applicable;  [35].
The MDR3 protein was primarily expressed in the liver, functioning as a floppase that translocated specifically phosphatidylcholine from the inner to the outer leaflet of the hepatocytes canalicular membranes [36]. Phosphatidylcholine was solubilized by canalicular bile salts to form mixed micelles, therefore protecting the biliary tree from exposure to toxic and detergent effects of bile salts [37]. The ABCB4 variants in this study impaired the floppase function of the MDR3 protein, and thus the depletion of phosphatidylcholine and elevation of hydrophobic bile acids in the biliary tubules damaged the integrity of the cholangiocyte membrane, leading to the development of intrahepatic cholestasis and presenting as hepatomegaly, pruritus, splenomegaly, jaundice and portal hypertension. Besides, low phosphatidylcholine levels would be expected to destabilize micelles and promote lithogenicity of bile with crystallization of cholesterol, which could facilitate liver damage by obstructing small bile ducts [38]. This could explain the occurrence of gallstones in a small number of children with PFIC3 as shown in Additional file 2: Table S1.
In this study, all PFIC3 patients exhibited increased serum GGT levels, constituting a biochemistry hallmark of this condition. Serum GGT was deemed to be mainly of hepatobiliary origin and has been used as a "liver test" for decades [39]. The reasons for elevated GGT values in those with hepatobiliary disease included de novo synthesis, release of membrane-bound GGT (by detergent effects of bile acids), regurgitation of bile into the blood stream, and change in permeability or destruction of biliary epithelial cells [40]. Due to the impaired MDR3 function, PFIC3 patients lacked phosphatidylcholine in the bile to form micelles, and thus the very detergent bile liberated GGT from the canalicular membrane, giving rise to cholangitis with high serum GGT activity [41,42].
At present, medical treatment was the first line of therapy offered to PFIC3 patient [9,43], and the major goal of medical treatment was to relieve symptoms, improve the nutritional status, and to treat or prevent complications due to cirrhosis and portal hypertension [38]. UDCA was the most common medicine in patients with PFIC3 [44], and the PFIC3 patients with residual phosphatidylcholine secretion and MDR3 expression, especially those with missense variants, responded to UDCA in 70% of cases [45], and even in those with cirrhosis, UDCA could delay PFIC3 progression [12]. In this study, 66.1% (39/59) of the patients had positive responses to oral UDCA, of whom 38.5% (15/39) fully recovered in terms of the laboratory changes. This was not surprising since UDCA had multiple mechanisms of action in cholestatic disorder including protection of cholangiocytes against cytotoxicity of hydrophobic bile acids, stimulation of hepatobiliary secretion of hydrophobic bile acids, inhibition of liver cell apoptosis, as well as anti-inflammation and immunomodulation [46,47].
Although most PFIC3 patients showed varying degrees of improvement in response to UDCA therapy, unfavorable prognosis was observed in some cases. Actually, this condition was progressive in the majority of affected patients, and carried a high risk of developing cirrhosis and liver failure during the first 2 decades of life [48]. In Fig. 3 The distribution of the 96 ABCB4 variants detected in the 95 PFIC3 patients in this study. The novel ABCB4 variants identified in this study were marked with asterisks. E2-E28 represented the 27 encoding ABCB4 exons this study, 41.1% (37/90) of PFIC3 patients had a poor prognosis, including 7 died, 27 having undergone while another 3 waiting for liver transplantation. On the last follow up, the condition remained stable in 53 patients (58.9%, 53/90), but their long-term prognoses were still uncertain, which needed to be followed up. So far, like other end-stage liver disease, liver transplantation remained the last resort in patients unresponsive to medical treatment [49]. Nevertheless, the lack of donor liver organ and lifelong burden of immunosuppressive therapy restricted the treatment option for this devastating condition [50].
ABCB4 variants exhibited remarkable heterogeneity, and the extent to which they impaired MDR3 floppase activity determined the course and outcome of the PFIC3 patients [21]. Depending on whether they affected the traffic, activity, or stability of the protein, ABCB4 variants could be classified as follows: (I) defective synthesis, mainly nonsense and frameshift variants, (II) affect protein maturation, (III) with little or no effect on protein maturation but defective proteins, (IV) affect the stability and (V) variants without detectable effects, providing the strong basis for the development of genotype-based therapies for PFIC3 [24]. This study found that PFIC3 patients with biallelic null variants exhibited earlier onset ages, lower UDCA response rate and more unpromising clinical outcomes, which clearly indicated that null ABCB4 variants were associated with severer PFIC3 phenotypes. Understanding the genotype-phenotype correlation contributed to the prediction of prognosis and provided additional guidance to physicians and patients about the likely disease course [15].

Conclusions
PFIC3 presented with hepatomegaly, pruritus, splenomegaly and jaundice with increased serum GGT level as a biochemistry hallmark. Although varying degrees of improvement in response to UDCA therapy were observed, 41.1% of PFIC3 patients exhibited unfavorable prognosis. ABCB4 genotypes of biallelic null variants were associated with severer PFIC3 phenotypes. Moreover, the 14 novel variants in this study expanded the ABCB4 variant mutation spectrum, and provided novel molecular biomarkers for the definite diagnosis of PFIC3 patients.